7.ProjectSummary/Abstract InacollaborativeresearchprojectthatIinitiatedbetweenthelabsofProf.PaulSondelandProf.Paul Harariduringmyresidencytraining,weexploredanovelapproachtoaugmentinganti-tumorimmuneresponse bycombiningtwoestablishedcancertreatments,radiationtherapy(RT)andtumor-specificantibodies(mAbs). Insingle-tumormurinemodelsofmelanoma,neuroblastoma,andheadandnecksquamouscellcarcinomawe observedacooperativeanti-tumorinteractionbetweenlocalRTandintratumoral(IT)injectionoftumor-specific mAbresulting,inpart,fromenhancedantibody-dependentcell-mediatedcytotoxicity.Tofurtheraugmentthis response,weinvestigatedcombinedRTandIT-immunocytokine(IC),afusion-proteinlinkingtumor-specific mAbtoIL2.Withthisweobservedagreateranti-tumorimmuneresponseresultingincompleteregressionof established(~5weekpostengraftment)tumorsinmostanimalsandamemoryTcellresponsethatrejected re-challengewithsimilartumorcells.ThisdemonstratedthatcombinedRT+IT-ICcanelicitapotent?in situ?tumorvaccineresponse.Tcellcheckpointblockadeisbecomingastandardofoncologiccarein certaincancersettingsandwethereforetestedthebenefitofaddinglocalRTandIT-ICtosystemiccheckpoint blockadeinourmurinemelanomamodel.Inmicebearinglargeprimarytumors(~7weekpostengraftment)or microscopicdistantsitesofdisease(IVinjectedonthedayofRT),thetriple-combinationofprimarytumorRT followedbyIT-ICinjectionofthistumorandsystemicTcellcheckpointblockadewithanti-cytotoxicT- lymphocyteantigen-4(CTLA-4)antibodyimprovedanimalsurvivalcomparedtocombinationsofanytwoof thesethreeinterventions.However,inthepresenceofestablished(~3-weekpostengraftment)distantsitesof diseasewehavenowobservedakeylimitationtoourinsituvaccinationstrategy.Inthepresenceofanun- radiatedsecondmacroscopictumor,thecombinationofprimarytumorRT+primarytumorIT-IC injectionisnomoreeffectivethanprimarytumorRTalone.Thissuggeststhatanuntreated,distant secondtumormaysuppressthegenerationofananti-tumorimmuneresponseintheprimarytumorfollowing RT+IT-IC.Wedescribethisas?concomitantimmunetolerance?(CIT). HereIhypothesizethat1)insituvaccinationmaybeachievedusinglocalRTandITinjectionoftumor- specificmAbwithIL2,2)regulatoryTcell(Tregs)harboredinanestablished,macroscopic,distanttumorsite mayexertCIT,and3)deliveringexternalbeamRT(EBRT)andIT-ICtoamouse?sprimarytumor,together withIV131I-NM404,amoleculartargetedradiotherapy(MTRT),willeliminateCITandenableaneffectiveinsitu vaccineresponsetoeradicatealltumorsinmicewithprimaryanddistantsitesofdisease. Iproposetotestthesehypothesesinmyindependentresearchlabusingestablishedtechniquesand animalmodelsthatIhaveusedtogeneraterobustpreliminarydata.Ihavesecuredthesupportofsix collaboratorswhoseexpertconsultationand/ormaterialassistancewillhelpensurethesuccessofthis researcheffort.Thisproposaldoesnotoverlapwithpriororongoingprojectsinthelabsofmyresearch mentors,whohaveexpressedtheircompletesupportformycareerdevelopment,myintellectual independence,andmypursuitoftheseresearchobjectivesintheindependentlabthatIwilllaunchinOctober. IhavepreviouslyappliedforthisDP5award(forRM-15-006)and,incomparisontothatpriorapplication, thecurrentproposalincludesmultiplerevisionsguidedbyreviewerfeedback.Thisapplicationalsoreflectsthe intervalpublicationofanadditionalfirst-authororiginalresearchmanuscriptthatisdirectlyrelatedtothe proposedresearch.Inanefforttoenhancethelevelofinnovationinthisproposal,Ihavecompletelyrevised Aim2and3.Aim2(previousAim2and3)nowspecificallyevaluatesthecriticalmechanisticunderpinningsof tumor-specificCITusingtransgenicmurinemodelstodeciphertheroleofTregsinthisnever-before-reported phenomenon.MeanwhileinAim3,IbuildfromourpreliminaryobservationthatCITcanbeovercomeby deliveringRTtothedistantsecondtumorsiteandhereIproposeanaltogetheruniqueapproachto circumventingCITbycombininganinsitutumorvaccinestrategywithasystemicmoleculartargeted radiotherapy(131I-NM404),whichmaysafelydeliverRTtoallsitesofmetastaticdiseasewithoutunduetoxicity orsystemicimmunesuppression.Inadditiontothesechangesspecificallyaimedatenhancingtheinnovation ofthisapplication,Ihavealsoincorporatedadditional,moreclinicallyrelevant,spontaneousandinducible transgenictumormodels(Aim1.3andthereafter).Thiswasdoneinanefforttofurtherstrengthenourrobust experimentalapproach.Tofurtherclarifythisapproach,Ihavealsomoreexplicitlyincorporatedastatistical planinthediscussionofeachsub-aim.WhilemypriorDP5proposalwasscoredquitefavorablyandadvanced tothefinalroundofinterviews,itismyhopethatwiththeserevisionsthecurrentapplicationmaywarrant funding.Thisawardwouldclearlyprovideatremendousaccelerationtomyindependentearlycareer developmentandthepaceatwhichmylabwillbeabletopursuetheexperimentsoutlinedherein.